Summary | Promise of epigenetic modulation as a target in atherosclerotic patients

August 26, 2017 - Prof. Erik Stroes (Amsterdam, The Netherlands)

Professor Stroes aimed to describe the overriding role of inflammatory activation in the process of atherogenesis, and how epigenetic targeting might counterbalance these effects. In all stages of the atherosclerotic process, from recruitment of monocytes through destabilization of plaque, inflammation plays a central role.

Degradation of the fibrous cap can result in a full occlusion of the vessel, and consequently MI. It has been shown that the ipsilateral side, where an inflamed plaque resides, gives rise to stroke, while the contralateral side did not show inflammation of the arterial wall.19 A more recent study using 18F-FDG PET imaging demonstrated that in the presence of risk factors for atherogenesis, and in case of overt atherosclerosis, an inflamed arterial wall is seen.20

CV patients can be characterized by a profound proinflammatory state that is observed at various levels; in the vessel wall, plasma and bone marrow.

Inflammatory cells do not only get activated once they arrive at the plaque, but activated monocytes are already found in the circulation of patients with elevated Lp(a). It has been described that the stickiness of their white blood cells is increased compared with controls.21 Even in bone marrow, increased cellular activity is seen in patients who were treated for several months following an ACS; hematopoietic stem and progenitor cells (HSPCs) of CVD patients had a higher progenitor potential than HSPCs of controls. This suggests that the bone marrow senses the ACS event and contributes to a prolonged inflammatory state.22 Thus, CV patients can be characterized by a profound proinflammatory state that is observed at various levels; in the vessel wall, plasma and bone marrow.

Now, can this process be targeted with therapy? The CANTOS trial evaluating treatment with an IL-1β antibody in post-ACS patients with elevated CRP levels as a marker of inflammation, has met its primary endpoint of reduction of MI, stroke and CV death. A related question is which effects the induced immunosuppression has on patients; in other words do we put patients at risk? This is where epigenetic therapy comes into play. Using an analogy of reducing fuel use in an airplane, Stroes explained that epigenetics refers to modifying, or tweaking, gene expression in a more subtle intermediate manner than turning expression on or off. In epigenetic processes, gene expression is modified without alteration of the genetic code itself. He illustrates this by using a movie as a metaphor of human life. Our DNA is the script, which is rigid, genetic regulation is the screen writing, and epigenetics is the director who will ultimately control and direct how the script is read.

Normally, DNA is tightly packed in chromosomes in the cell nucleus, which makes it hardly accessible for transcription factors. The DNA is wrapped around protein complexes called nucleosomes, which consist of various histone proteins. This level of organization is referred to as chromatin. These histones have protruding tails that can be chemically modified. Modification of the DNA or the histones can result in local opening of the chromatin structure, which makes the DNA more accessible for transcription factors. Similarly, the chromatin can be compacted, which generally results in gene silencing.

Various types of histone modifications can take place, including methylation and acetylation. Multiple enzymes are involved in placing or taking away these histone marks, therefore sometimes referred to as ‘writers’ and ‘erasers’, thereby regulating gene transcription. Depending on the location, histone methylation can lead to gene activation or repression, while histone acetylation always results in activation of gene transcription by opening up the chromatin, allowing access of transcription factors.

In advanced human plaques, many histone modifications have been described. An overrepresentation of activating methylation marks is seen, and a decrease of repressing methylation marks. This might be related to the activated monocytes seen in patients with elevated Lp(a).21 Indeed, increased trimethylation of histone 3 at lysine 4 (H3K4KMe3), a mark associated with open chromatin, is seen in the promoter regions of genes that encode proinflammatory cytokines (unpublished data, Van der Valk et al.). The histone modifications can in turn be recognized by the so-called epigenetic readers, among which is the family of bromodomain and extra-terminal domain (BET) proteins. Multiple BET proteins exist, of which BRD4 is the most important one. BRD4 binds DNA polymerase. Inhibiting the BET protein BRD4 will inhibit DNA transcription into mRNA. This has a variety of downstream effects, and BET inhibition has received a lot of attention in oncology.

Focusing on the inflammation cascade, it has been shown that inhibiting BET proteins with siRNA in epithelial cells leads to inhibition of adhesion of monocytes to the endothelium.24

The rationale of epigenetic modulation is to reduce ‘redundant’ inflammation, without risking overall immune suppression.

Apabetalone (or RVX-208) is a selective BD2 inhibitor. BD2, together with a BET protein, normally recognizes acetylated histone tails. When apabetalone binds BD2, it prevents recognition of the histone mark, thereby preventing gene transcription. Treating endothelial cells with apabetalone results in inhibition of expression of the VCAM-1 adhesion molecule and in lower adhesiveness of monocytes to the endothelial cells (Resverlogix, data on file). Moreover, adding a BET inhibitor to macrophages in vitro has been shown to decrease their proinflammatory activity.25

It has been studied in mice whether these effects translate into a decrease of atherogenesis. Indeed, after treatment with apabetalone, hyperlipidemic ApoE-/- mice showed a 40% reduction of atherogenesis.26

When apabetalone is added to human peripheral blood monocytes from healthy volunteers, downregulation of a wide array of proinflammatory cytokines and chemokines and their receptors is seen within 24 hours (Resverlogix, data on file), which suggests a possible anti-inflammatory potential in humans. The safety data of for example the ASSURE study, suggest a decreased proinflammatory state in CVD patients treated with apabetalone. For instance, CRP is lowered by about 21% and TNF by 14%.27

Thus, these experimental findings might suggest that epigenetic therapy by means of BET inhibition may reduce inflammation, and potentially thereby reduce atherogenesis. The rationale of epigenetic modulation is to reduce ‘redundant’ inflammation, without risking overall immune suppression. The first safety data in human patients are reassuring.